Tissue-Like 3D Standard and Protocols for Microscope Quality Management

Abrams, Benjamin, Pengo, Thomas, Wee, Tse-Luen, Deagle, Rebecca C, Vuillemin, Nelly, Callahan, Linda M, Smith, Megan A, Kubow, Kristopher E, Girard, Anne-Marie, Rappoport, Joshua Z, Bayles, Carol J, Cameron, Lisa A, Cole, Richard, Brown, Claire M (April 2023) Tissue-Like 3D Standard and Protocols for Microscope Quality Management. Microscopy and Microanalysis, 29 (2). pp. 616-634. ISSN 1431-9276

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Abstract

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

Item Type:	Paper
Subjects:	Investigative techniques and equipment Investigative techniques and equipment > microscopy > flourescence microscopy Investigative techniques and equipment > microscopy
CSHL Authors:	Wee, Tse-Luen
Communities:	CSHL Cancer Center Shared Resources > Microscopy Service CSHL labs > Spector lab CSHL labs > Wee Lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	5 April 2023
Date Deposited:	04 Jan 2024 19:09
Last Modified:	08 Feb 2024 20:12
PMCID:	PMC10617369
Related URLs:	Publisher
URI:	https://repository.cshl.edu/id/eprint/41377

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